During terminal differentiation epidermal keratinocytes manifest a programmed set of morphological and biochemical changes that result in the production of two major structures: 1) an envelope of covalently linked protein enclosing 2) a constellation of keratin intermediate filaments. A major precursor of the envelope is a 46 kDa polypeptide called involucrin which is incorporated into the envelope by a calcium-dependent transglutaminase. Involucrin is likely to account for the majority of glutamyl-lysine linkages that hold the envelope together. In spite of its importance, no information is available regarding which glutamines within involucrin are targeted for crosslinking by transglutaminase or which sections of the involucrin molecule are essential for high strength envelope formation. Active envelope formation is essential for survival and abnormal envelope formation is a feature of several epidermal diseases. The ultimate goal of the experiments described in this proposal is to understand the role of involucrin in the envelope assembly process and how this impacts on the disease state. To provide a tool for these studies, we cloned and sequenced the complete human involucrin gene. Our sequence revealed that the molecule is an extended alpha-helix composed of highly similar, tandemly linked repeats of ten amino acids. Each repeat contains three glutamine residues, each of which is a potential crosslink site. In the present experiments we propose to utilize a unique approach to study envelope formation and express the cloned involucrin gene in a variety of cell types to gain new knowledge regarding 1) which glutamine residues within the involucrin peptide are the substrates for the calcium-dependent transglutaminase, 2) whether involucrin can participate in envelope formation in rat keratinocytes and 3) to determine the role of involucrin in stabilizing the cornified envelope.